Activation of the Gi protein-coupled A3 adenosine receptor (AR) is associated with anticancer, antiischemic and antiinflammatory effects, and clinical trials of several prototypical selective agonists are underway. See, for example, Ochaion, A., et al., Cell Immunol. 2009, 258:115-122; and Cohen, S., et al., J. Cell. Physiol. 2011, 226: 2438-2447. A3AR antagonists are being examined as promising agents for the treatment of glaucoma. Wang, Z., et al., Exp. Eye Res. 2010, 90:146-154; and Okamura, T., et al., Bioorg. Med. Chem. Lett. 2004, 14: 3775-3779. Characterization of the A3AR in tissues and assays to establish a structure-activity relationship (SAR) of newly synthesized compounds for drug discovery, as for many other G protein-coupled receptors (GPCRs), is often dependent on the use of high affinity radioligands. Fredholm, B. B., et al., Pharmacol Rev 2011; 63:1-34. Fluorescent agonists and antagonists of GPCRs have been studied as molecular probes for binding experiments. Middleton R. J., et al., Curr Opin Chem Biol 2005; 9:517-25; Kuder, K., et al., Curr. Med. Chem. 2008; 15:2132-43; Bajaj, A., et al., Biochemistry 2004; 43:13564-13578. Recently, several 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivatives have been reported as fluorescent ligands of the ARs, including the A3AR. Cordeaux, Y., et al., FASEB J 2008; 22:850-60; Corriden, R., et al., Proc. Brit. Pharmacol. Soc., 2009, vol. 7, Abstract 086P. http://www.pA2online.org/abstracts/Vol7Issue4abst086P.pdf, and used to study receptor complexes. However, many of these fluorescent tracers are relatively nonselective within the AR family.
Receptor-selective fluorescent ligands are used increasingly as tools for the study of receptor physiology and pathophysiology at the cellular and even the subcellular level; Kuder et al., infra. Furthermore, they are being increasingly investigated as tools in drug discovery research; Middleton, R. J., et al., infra. In both cases, techniques employing receptor-selective fluorescent ligands have proved to be complementary to, and in several cases even superior to, the traditional radioligand-based techniques. Increasing costs and public concerns associated with radioactive isotope handling and disposal are also making the use of fluorescent ligands more attractive in research and diagnostics. With the increasing importance of GPCRs in pharmacology and the search for newer, more potent and/or selective receptor ligands and drugs, there is an accompanying need for the design and development of novel highly potent and selective fluorescent ligands. Such ligands will likely aid in the investigation of the physiological and pathophysiological functions of GPCRs and also in the development of drugs acting specifically at these receptors.
The foregoing shows that there exists an unmet need for fluorescent ligands which are selective antagonists of the A3 adenosine receptor.